Hemostasis is a naturally occurring process which results in the spontaneous arrest of bleeding from damaged blood vessels. For example, precapillary vessels will contract immediately when an individual is cut. Within seconds after such a cut, the process of hemostasis begins. At a site of injury with disruption of a blood vessel or exposure of subendothelial vascular tissue, two events rapidly occur. The two limbs of the hemostatic system, each comprised of many molecules, are activated. The coagulation (clotting) system is immediately initiated producing thrombin; and blood platelets adhere to matrix proteins. The platelets are activated, in part by thrombin, and release adenosine diphosphate ("ADP") leading to aggregation of additional platelets into a growing platelet plug in concert with the conversion of fibrinogen in the blood to the insoluble fibrin gel. This hemostatic plug is strengthened by additional enzymatic cross-linking. Over time it is dissolved during tissue repair to result in normal tissue and blood vessel, with or without residual pathology of the local vessel wall or tissue.
Thrombogenesis is an altered, pathogenic state of one or both limbs of the hemostatic system. In such states, an intravascular (arterial or venous) thrombus results from a pathological disturbance of hemostasis. A platelet-rich thrombus, for example, is thought to be initiated by the adhesion of circulating platelets to the wall of an arterial vessel. This initial adhesion, activation by thrombin or other agonists, and the concomitant release of ADP from platelets, is followed by platelet-platelet interaction or aggregation. Fibrin formation is associated with the platelet thrombus but is a minor component. The arterial thrombus can grow to occlusive proportions in areas of slower blood flow.
In contrast, fibrin-predominant thrombi develop initially in areas of stasis or slow blood flow in blood vessels and may resemble a blood clot formed in vitro. The bulk of venous thrombi comprise a fibrin network enmeshed with red blood cells and platelets. A venous thrombus can-establish a "tail" that can detach and result in embolization of the pulmonary arteries. Thus, it will be understood that arterial thrombi cause serious disease by local ischemia, whereas venous thrombi do so primarily by distant embolization.
A platelet plug formed solely by ADP-stimulating platelet interaction is unstable. Immediately after the initial aggregation and viscous metamorphosis of platelets, as noted above, fibrin becomes a constituent of a platelet-rich thrombus. Production of thrombin occurs by activation of the reactions of blood coagulation at the site of the platelet mass. This thrombin may activate the initial adherent platelets and stimulates further platelet aggregations. Platelet aggregation is stimulated not only by inducing the release of ADP from the platelets, but also by stimulating the synthesis of prostaglandins, which as aggregating agents are more powerful than ADP, and by the assembly of the prothrombinase complex on the activated platelets to accelerate the formation of more thrombin, the very powerful activator of platelets.
The coagulation of blood results in the formation of fibrin. It involves the interaction of more than a dozen proteins in a cascading series of proteolytic reactions. At each step a clotting factor zymogen undergoes limited proteolysis and itself becomes an active protease. This clotting-factor enzyme activates the next clotting factor zymogen until thrombin is formed which connects fibrinogen to the insoluble fibrin clot. The blood clotting factors include factor I (fibrinogen), factor II (prothrombin), tissue factor (formerly known as factor III), factor IV (Ca.sup.2+), factor V (labile factors), factor VII (proconvertin), factor VIII (antihemophilic globulin, or "AHG"), factor IX (Christmas factor), factor X (Stuart factor), factor XI (plasma thromboplastin antecedent, or "PTA"), factor XII (Hageman factor), factor XIII (fibrin-stabilizing factor), and factors HMW-K (high-molecular-weight kininogen, or Fitzgerald factor), PRE-K (prekallikrein, or Fletcher factor), Ka (kallikrein), and PL (phospholipid).
Fibrinogen is a substrate for the enzyme thrombin (factor IIa), a protease that is formed during the coagulation process by the activation of a circulating zymogen, prothrombin (factor II). Prothrombin is converted to the active enzyme thrombin by activated factor X in the presence of activated factor V, Ca.sup.2+, and phospholipid.
Two separate pathways, called the "intrinsic" and "extrinsic" systems, lead to the formation of activated factor X. In the intrinsic system, all the protein factors necessary for coagulation are present in the circulating blood. In the extrinsic system, tissue factor, which is not present in the circulating blood, is expressed on damaged endothelium, on activated monocytes by cells in the arteriosclerotic plaque or by cells outside the vessel wall. Tissue factor then acts as the receptor and essential cofactor for the binding of factor VII resulting in a bimolecular enzyme to initiate the extrinsic pathway of coagulation. This mechanism also activates the intrinsic pathway of coagulation. The tissue factor pathway can very rapidly clot blood.
Blood can also be clotted by the contact system via the intrinsic pathway of coagulation. The mechanism is somewhat slower than the tissue factor pathways, presumably because of the larger number of reactions that are required. Both the intrinsic system and extrinsic system pathways must be intact for adequate hemostasis. See Zwaal, R. F. A., and Hemker, H. C. "Blood cell membranes and hemostasis." Haemostasis, 11:12-39 (1982).
Thrombosis and a variety of related forms of diseases are associated with, and result from, activation of one or more of the coagulation protease cascades pathways, and disorders of regulation of the combined coagulation/anticoagulation/fibrinolytic pathways. These diseases affect approximately 2.5 million individuals annually in the United States. Some three percent of the U.S. population over the age of 45 develop some form of thrombotic disease or disseminated coagulation each year. Other thrombotic diseases are hereditary and may affect 100,000 people annually. Seventy percent of such diseases are fatal by 45 years of age.
Of acquired thrombotic diseases, coronary thrombosis at about 1.5 million cases per year, pulmonary thromboembolism at about 400,000 cases per year and severe septic shock at more than 300,000 cases per year, disseminated intravascular coagulation (DIC) at about 350,000 cases per year, and deep vein thrombosis at about 175,000 cases per year, predominate. However, diseases such as menigococemia, hemorrhagic fever virus infections, and a variety of other diseases produce significant morbidity and mortality as well. See, e.g., Kaplan, K. "Coagulation Proteins in Thrombosis." In Hemostasis and Thrombosis, Colman, R. W., et al. eds., pages 1098 et seq. (2d Ed. J. B. Lippincott Co. 1987). Some of the most acutely severe forms of disseminated intravascular coagulation affect children secondary to a variety of infectious diseases. Current treatment for thromboembolic disease is by no means satisfactory, and includes the use of anticoagulants, antithrombotic drugs and thrombolytic agents.
One of the most well-known anticoagulants is heparin. Discovered in 1922, heparin is a heterogenous group of straight-chain anionic mucopolysaccharides, called glycosaminoglycans, of molecular weights that average 15,000 daltons. Commercial heparin typically consists of polymers of two repeating disaccharide units: D-glucosamine-L-iduronic acid and D-glucosamine-D-glucuronic acid. It is typically prepared from both bovine lung and porcine intestinal mucosa, and has also been obtained from sheep and whales.
While heparin occurs intracellularly in mammalian tissues that contain mast cells, it is limited to a macromolecular form of at least 750,000 daltons. Furthermore, this heparin has only 10-20% of the anticoagulant activity of commercial heparin. Heparan sulfate, a compound similar to heparin but with less anticoagulant activity is a ubiquitous component at the mammalian cell surface. When native heparin is released from its bound and inactive state in the metachromatic granules of mast cells, it is ingested and rapidly destroyed by macrophages. Heparin cannot be detected in the circulating blood.
When injected intravenously, commercially prepared heparin impairs blood coagulation. It acts by complexing with antithrombin III, a serine protease inhibitor that neutralizes several activated clotting factors, i.e., factors XIIa, kallikrein (activated Fletcher factor), XIa, IX, Xa and thrombin (IIa). However, it is most active in inhibiting free thrombin and activated factor X (Xa). Although antithrombin III was thought to be the only macromolecule able to inactivate thrombin, other plasma proteins are now known to possess this activity. Antithrombin III can form irreversible complexes with serine proteases, and, as a result, the above protein factors are inactivated. Griffith, M. J. "Heparin-Catalyzed Inhibitors/Protease Reactions: Kinetic Evidence for a Common Mechanism of Action of Heparin," Proc. Natl Acad Sci USA, 80:5460-5464 (1983). Heparin markedly accelerates the velocity, although not the extent of this reaction. A ternary complex is apparently formed between heparin, antithrombin III, and the clotting factors. Bjork, I., and Lindahl, U. "Mechanism of the Anticoagulant Action of Heparin" Mol. Cell. Biochem., 48:161-182 (1982). Low concentrations of heparin increase the activity of antithrombin III, particularly against factor Xa and thrombin and this forms the basis for the administration of low doses of heparin as a therapeutic regimen.
While purified commercial preparations of heparin are relatively non-toxic, a chief complication of therapy with heparin is hemorrhage. Heparin also causes transient mild thrombocytopenia in about 25% of the patients, severe thrombocytopenia in a few, and occasional arterial thrombi. The mild reactions result from heparin-induced platelet aggregation, while severe thrombocytopenia follows the formation of hepar-independent antiplatelet antibodies complexes. It is to be understood that, in all patients given heparin, platelet counts must be monitored frequently, any new thrombi might be the result of the heparin therapy, thrombocytopenia sufficient to cause hemorrhage should be considered to be heparin-induced, and that thrombosis thought to result from heparin should be treated by discontinuation and substitution of an agent that inhibits platelet aggregation and/or an oral anticoagulant.
Severe thrombocytopenia, hemorrhage, and death have occurred even in patients receiving "low-dose" heparin therapy. Heparin therapy is, furthermore, contraindicated in patients who consume large amounts of ethanol, who are sensitive to the drug, who are actively bleeding, or who have hemophilia, purpura, thrombocytopenia, intracranial hemorrhage, bacterial endocarditis, active tuberculosis, increased capillary permeability, all sorts of lesions of the gastrointestinal tract, severe hypertension, threatened abortion, or visceral carcinoma. Furthermore, heparin is to be withheld during and after surgery of the brain, eye, or spinal cord, and is not to be administered to patients undergoing lumbar puncture or regional anesthetic block. Goodman and Gillman's The Pharmacological Basis of Therapeutics, pages 1339-1344 (7th ed. 1985).
There are a number of oral anticoagulants that are also available for clinical use. Many anticoagulant drugs have been synthesized as derivatives of 4-hydroxycoumarin or of the related compound, idan-1,3-dione. The essential chemical characteristics of the coumarin derivatives for anticoagulant activity are an intact 4-hydroxycoumarin residue with a carbon constituent at the 3 position. There are a number of differences in the pharmacokinetic properties and toxicities of these agents, however, and racemic warfarin sodium is the most widely used oral anticoagulant in the United States.
The major pharmacological effect of oral anticoagulants is inhibition of blood clotting by interference with the hepatic post translational modification of the vitamin K-dependent proteins among which are the clotting factors, i.e., Factors II, VII, IX and X. These drugs are often called indirect anticoagulants because they act only in vivo, whereas heparin is termed a direct anticoagulant because it acts in vitro as well. Again, hemorrhage is the main unwanted effect caused by therapy with oral anticoagulants, and such therapy must always be monitored. In reported order of decreasing frequency, complications include ecchymoses, hematuria, uterine bleeding, melena or hematochezia, epistaxis, hematoma, gingival bleeding, hemoptysis, and hematemesis. All of the contraindications described above in regard to the use of heparin apply to the anticoagulants as well.
Anti-platelet drugs suppress platelet function and are used primarily for arterial thrombotic disease, whereas anticoagulant drugs, such as warfarin and heparin suppress the synthesis or function of clotting factors and are used to control venous thromboembolic disorders. There are a number of anti-platelet drugs, the most well-known being aspirin. The efficacy of these agents for acute treatment has, however, not been established and there is a real problem with aspirin hemorage.
Thrombolytic drugs include streptokinase, urokinase, tissue plasminogen activator, and APSAC (acylated plasminogen streptokinase complex). These are proteins which have demonstrated efficacy for the treatment of acute thrombotic disease. They promote the dissolution of thrombi by stimulating the conversion of endogenous plasminogen to plasmin, a proteolytic enzyme that hydrolyzes fibrin. The use of these agents is limited, however, to acute thrombotic disease. Fibrinolytic agents are used primarily for the treatment of patients with established coronary arterial thrombosis.
Effective therapy for a variety of forms of intravascular activation of the coagulation protease cascades, whether thrombosis or the more catastrophic forms such as those associated with vasomotor collapse (septic shock) and other forms of disseminated intravascular coagulations are not entirely satisfactory, and in the case of septic shock is entirely unsatisfactory. The need for effective therapy that is capable of rapidly arresting arterial thrombogenesis is recognized as an important therapeutic deficiency. This is evident from the recent evidence that heparin is entirely ineffective in preventing rethrombosis of the 11-20% of patients that rethrombose at the completion of thrombolytic therapy with tissue plasminogen activator.
The present invention was made in response to these needs and relates to antagonists of factor VII and specific antagonists of the procoagulant activity of factor VIIa and the tissue factor:factor VIIa complex. The invention includes monoclonal-type antibodies produced by cell systems including bacteria, such as E. coli, or by hybrid cell lines, characterized in that the antibodies, or functional fragments thereof, have predetermined specificity to factor VII, to factor VIIa, and/or to the bimolecular complex of tissue factor and factor VIIa, are effective for neutralization of these targets, and find application as antithrombotic agents for syndromes such as disseminated intravascular coagulation ("DIC") and venous thrombosis. The present invention also relates to the use of these monoclonal-type antibodies in methods for the purification of factor VII, factor VIIa and the bimolecular complex referred to above, and in methods for the immunoassay or immunodetection of factor VII, factor VIIa and the tissue factor/factor VIIa bimolecular complex. The purification of factor VII, factor VIIa and the tissue factor/factor VIIa complex from a biological sample containing these antigens can be carried out by immuno-affinity chromatography in which the biological sample is passed through an immunoadsorbant column or slurry comprising the novel monoclonal-type antibodies or antibody fragments of this invention bound to a solid base support to thereby selectively adsorb said antigenic targets. The immunoassay of factor VII, factor VIIa and the tissue factor/factor VIIa bimolecular complex for determining the presence or concentration of these target antigens in a biological sample containing them can be carried out by contacting said sample with a known amount of the novel monoclonal-type antibody of this invention and measuring the resulting adsorbed monoclonal antibody.
Factor VII is a vitamin K-dependent zymogen of the active serine protease VIIa. Factor VII functions to form a complex with tissue factor in blood, and on conversion to VIIa forms the complex which then activates factor X by converting factor X to factor Xa. Procoagulant activity is only associated with the tissue factor:VIIa complex. Free factor VII and free factor VIIa, as well as the tissue factor:factor VII complex, do not possess procoagulant activity. Factor VII is a single polypeptide chain of about 50,000 daltons that can, in a purified system, be activated by proteolytic cleavage of disulfide bonds by factor Xa, factor IXa, thrombin and factor XIIa. Takase, T. et al., "Monoclonal Antibodies to Human Factor VII: A One Step Immunoradiometric Assay for VIIag, J. Clin. Pathol., 41:337-341 (1988). Human factor VII, when partially or completely activated, yields a protein comprised of two polypeptide chains linked by disulfide bridges. Factor VII and VIIa may be used interchangeably in this document and will be designated VII/VIIa when target interchangeability is to be indicated.
With the advent of hybridoma technology first developed by Kohler and Milstein, it is now possible to attempt to generate monoclonal antibodies which are essentially homogenous compositions having uniform affinity for a particular binding site. The production of mouse hybridomas by these investigators is described in Nature, 256:495-497 (1975) and Eur. J. Immunol., 6:511-519 (1976). Further procedures are described in Harlow, E., and Lane D., "Antibodies: A Laboratory Manual" (Cold Spring Harbor Laboratory 1988). According to the hybridoma method, tissue-culture adapted mouse myelomas cells are fused to spleen cells from immunized mice to obtain the hybrid cells, called "hybridomas," that produce large amounts of a single antibody molecule. Generally, animals are injected with an antigen preparation, and if an appropriate humoral response has appeared in the immunized animal, an appropriate screening procedure is developed. The sera from test bleeds of the immunized animal are used to develop and validate the screening procedure, and after an effective screen has been established, the actual production of hybridomas is begun. Several days prior to the fusion, which is generally carried out in the presence of polyethylene glycol as described by Galfe et al. Nature, 266:550-552 (1977), followed by selection in HAT medium (hypoxanthine, aminopterin and thymidine) as described by Littlefield, Science, 145:709-710 (1964), animals are boosted with a sample of the antigen preparation. For the fusion, antibody secreting cells are prepared from the immunized animal, mixed with the myeloma cells, and fused. After the fusion, cells are diluted in selective medium and plated in multi-welled culture dishes. Hybridomas may be ready to test as soon as about one week after the fusion, but this is not certain. Cells from positive wells are grown, subcloned, and then single-cells are cloned.
It is understood that hybridoma production seldom takes less than two months from start to finish, and can take well over a year. The production of monoclonal antibodies has been described in three stages: (1) immunizing animals (2) developing the screening procedure and (3) producing hybridomas. It is also understood that any one of these stages might proceed very quickly but that all have inherent problems. For example, while immunization can be carried out with virtually any foreign antigen of interest, many difficulties arise and variations may be required for any specific case in order to generate the desired monoclonal antibodies. Prior to attempting to prepare a given hybridoma, there is no assurance that the desired hybridoma will be obtained, that it will produce antibody if obtained, or that the antibody so produced will have the desired specificity or characteristics. Harlow, E., and Lane, D., supra at Chapter 6.
The production of monoclonal antibodies to human factor VII has been reported, and these reagents are said to have been used to make immunodepleted plasma or to detect factor VII cross reactive material in factor VII deficient patients. Id. The production of monoclonal antibodies to factor VII for their use in a one step, immunoradiometric assay for factor VII:ag has also been reported. Id. The authors reported the preparation of three mouse monoclonal antibodies, two of which were said to bind, to factor VII:ag, and two of which were said to be inhibitors of factor VII in vitro. See also Howard et al., J. Clin. Chem., 35:1161 (1989). No monoclonal antibodies against either factor VII or factor VIIa have been described which therapeutically interfere with the binding of factor VIIa to tissue factor or which neutralize the activity of the tissue factor/factor VIIa complex.